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Journal of Ethnopharmacology 115 (2008) 238–248

Repression of calcitonin gene-related peptide expressionin trigeminal neurons by a Theobroma cacao extract�

Marcie J. Abbey, Vinit V. Patil, Carrie V. Vause, Paul L. Durham ∗Department of Biology, Missouri State University, 901 S. National Avenue, Springfield, MO 65897, USA

Received 17 April 2007; received in revised form 17 September 2007; accepted 25 September 2007Available online 5 October 2007

bstract

thnopharmacological relevance: Cocoa bean preparations were first used by the ancient Maya and Aztec civilizations of South America toreat a variety of medical ailments involving the cardiovascular, gastrointestinal, and nervous systems. Diets rich in foods containing abundantolyphenols, as found in cocoa, underlie the protective effects reported in chronic inflammatory diseases. Release of calcitonin gene-related peptideCGRP) from trigeminal nerves promotes inflammation in peripheral tissues and nociception.im of the study: To determine whether a methanol extract of Theobroma cacao L. (Sterculiaceae) beans enriched for polyphenols could inhibitGRP expression, both an in vitro and an in vivo approach was taken.esults: Treatment of rat trigeminal ganglia cultures with depolarizing stimuli caused a significant increase in CGRP release that was repressedy pretreatment with Theobroma cacao extract. Pretreatment with Theobroma cacao was also shown to block the KCl- and capsaicin-stimulatedncreases in intracellular calcium. Next, the effects of Theobroma cacao on CGRP levels were determined using an in vivo model of temporo-

andibular joint (TMJ) inflammation. Capsaicin injection into the TMJ capsule caused an ipsilateral decrease in CGRP levels. Theobroma cacao

xtract injected into the TMJ capsule 24 h prior to capsaicin treatment repressed the stimulatory effects of capsaicin.onclusions: Our results demonstrate that Theobroma cacao extract can repress stimulated CGRP release by a mechanism that likely involveslockage of calcium channel activity. Furthermore, our findings suggest that the beneficial effects of diets rich in cocoa may include suppressionf sensory trigeminal nerve activation.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Calcitonin gene-related peptide; Trigeminal; Inflammation; Tempor

. Introduction

The trigeminal nerve is the primary sensory nerve of the facend head that connects the peripheral tissues and the central ner-ous system (O’Conner and Van der Kooy, 1986). Trigeminalerves are responsible for the perception of touch, temperature,nd nociception, and transmission of this information to the cellody in the trigeminal ganglion and trigeminal nucleus caudalisn the brain. Activation of trigeminal nerves, and the subsequent

eripheral and central release of neuropeptides, is involved inhe underlying pathology of chronic painful inflammatory condi-ions such as migraine (Buzzi, 2001; Pietrobon and Striessnig,

� This work was supported by NIH grants DE015385 and DE017805.∗ Corresponding author at: Department of Biology, 225 Temple Hall, Missouritate University, Springfield, MO 65897, United States. Tel.: +1 417 836 4869;ax: +1 417 836 4204.

E-mail address: pauldurham@missouristate.edu (P.L. Durham).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.09.028

dibular joint; Polyphenols; Vanilloid receptor

003) and temporomandibular joint disorders (TMJD) (Kopp,001).

Calcitonin gene-related peptide (CGRP) is an abundantolypeptide widely expressed in both central and peripheral neu-ons, including trigeminal ganglion neurons. CGRP is knowno promote inflammation and nociception (van Rossum et al.,997). Elevated levels of CGRP have been reported to cor-elate with pain severity during migraine attacks (Buzzi and

oskowitz, 1992; Edvinsson and Goadsby, 1994) and TMJDHolmlund et al., 1991; Appelgren et al., 1993). TMJD is alinical term used to describe the number of related disordersffecting the jaw joint, masticatory muscle and associated struc-ures (Schiffman et al., 1990). About 60–70% of the generalopulation has at least one symptom of TMJD, defined as pain

r limited joint opening (Dimitroulis, 1998). Currently, there areo FDA approved pharmacological treatments for TMJD.

Cocoa bean preparations were first used by the ancient Mayand Aztec civilizations of South America to treat a variety of

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M.J. Abbey et al. / Journal of Eth

edical ailments involving the cardiovascular, gastrointestinal,nd nervous systems (Dillinger et al., 2000). In addition, cocoaas documented to be beneficial in the treatment of general

nd abdominal pain, toothache, and pain associated withoints (rheumatism). Thus, a therapeutic benefit of cocoa as a

edical treatment appears to involve decreasing sensory nervectivation. Recently, phenolic phytochemicals have receivedrowing scientific interest in an effort to explain some ofhe health benefits associated with fruit- and vegetable-richiets. While polyphenols occur abundantly in teas, grapes, anderries, higher levels of total phenolics are reported in cocoaeans (Sanbongi et al., 1998; Lee et al., 2003). Polyphenolsisplay a wide range of biochemical and pharmacologicalctivities that strongly suggest a role in promoting health andreventing disease (Yoon and Baek, 2005). Recent efforts tonderstand the biological effects of polyphenols have primarilyocused on modulation of immune and endothelial cell functionMiddleton and Kandaswami, 1992; Stoclet et al., 2004;

illiams et al., 2004). However, regulation of sensory neuronshat mediate neurogenic inflammation and pain transmission byocoa polyphenols has not been directly investigated. Thus, theoal of this study was to investigate the effect of a Theobromaacao L. (Sterculiaceae) bean extract enriched for polyphenolsn CGRP expression using trigeminal ganglia cultures and ann vivo model of TMJ inflammation.

. Materials and methods

.1. Preparation of Theobroma cacao bean extract

Organic raw Ecuadorian cacao beans, Theobroma cacao,ere obtained from Nature’s First Law (San Diego, CA) and

tored at 4 ◦C. Beans (4 g), outer skin intact, were roasted for0 min at 177 ◦C, finely crushed after cooling, homogenizedmin in 30 ml of methanol at room temperature, and then mixedigorously for 1 min. Following centrifugation at 3000 × g formin, 10 ml of the supernatant was dispensed to a 100 mm Petriish and concentrated by evaporation at room temperature toml. To remove remaining particulate matter, the concentrated

iquid was centrifuged at 3000 × g for 5 min and the supernatantas transferred to a new tube. The concentrated methanol extractas stored at −20 ◦C and will be referred to as Theobroma cacao

xtract. The dry weight yield was 17 �g/g (w/w) of cocoa beans.he average total phenolic content of the extract (25.5 mg/ml;= 3) expressed as gallic acid equivalents (GAEs) was deter-ined using the Folin–Ciocalteu method (Singleton and Rossi,

965; Lee et al., 2003). Briefly, 0.25 N Folin–Ciocalteu reagentas added to the diluted sample or gallic acid, the samplesixed, and incubated for 3 min at room temperature. Sodium

arbonate (1 N) was added to each sample and after mixing theamples were incubated for 7 min. After addition of water, theamples were again mixed and then incubated for 2 h prior toeasurement of absorbance at 760 nm.

.2. Animals

Neonatal Sprague Dawley rats (Charles River Laboratoriesnc., Wilmington, MA) were used in all in vitro experiments

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rmacology 115 (2008) 238–248 239

hile adult female post-estrous Sprague Dawley rats were uti-ized for the in vivo studies. Animals were housed in clean plasticages on a 12-h light/dark cycle with unrestricted access to foodnd water. All animal care and procedures were conducted inccordance with institutional and National Institutes of Healthuidelines.

.3. Primary cultures of trigeminal neurons

Primary cultures of trigeminal ganglia were established basedn our previously published protocols (Durham and Russo,999; Bowen et al., 2006). Briefly, cells from ganglia isolatedrom 3–5-day-old rats were resuspended in L15 medium con-aining 10% fetal bovine serum (Atlanta Biologicals, Norcross,A), 50 mM glucose, 250 �M ascorbic acid, 8 �M glutathione,mM glutamine, and 10 ng/ml mouse 2.5 S nerve growth

actor (Alomone Laboratories, Jerusalem, Israel). Penicillin100 units/ml), streptomycin (100 �g/ml), and amphotericin B2.5 �g/ml, Sigma) were also added to the supplemented L15edia, which will be referred to as L15 complete medium.or secretion studies, dissociated cells from the equivalent of4 ganglia were plated on 24-well poly-d-lysine (PDL)-coatedissue culture plates (BD Biosciences, Bedford, MA) and incu-ated at 37 ◦C at ambient CO2. For the calcium measurements,rigeminal cultures were enriched in neuronal cells (>90%) byensity-gradient centrifugation as previously described (Bowent al., 2006). Briefly, after dissociation, the cell pellet wasesuspended in 3 ml plating medium containing 1 mg/ml bovineerum albumin (BSA). Cells were carefully layered onto 6 mlf plating medium containing 10 mg/ml BSA in a 15 ml coni-al tube and centrifuged at 100 × g for 3 min. The pellet wasesuspended in L15 complete medium and plated at a density of.5–2 ganglia per well in a 24-well plate, which corresponds to0,000–65,000 cells per well.

.4. CGRP secretion assay

Trigeminal ganglion primary cultures maintained for 48 h in15 complete medium were incubated in 250 �l per well ofEPES-buffered saline (HBS; 22.5 mM HEPES, 135 mM NaCl,.5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 3.3 mM glucose,nd 0.1% BSA, pH 7.4). The amount of CGRP released into theBS medium was determined using a CGRP-specific radioim-unoassay (Bachem/Peninsula Laboratories Inc., San Carlos,A). In each experiment, the basal (unstimulated) amount ofGRP secreted into the medium in 1 h was determined and wassed to normalize for differences between the wells.

The effects of 90 mM KCl or 2 �M 8-methyl-N-vanillyl--nonenamide (capsaicin; Sigma, St. Louis, MO) on CGRPecretion was determined by incubating the cells for 1 h inBS and the amount of CGRP measured. For inhibition stud-

es, cultures were pretreated for 30 min with 1.7, 8.5, 17, or4 �g/g of extract dry weight, which corresponds to 10, 50,

00, or 200 �g/ml GAE of Theobroma cacao extract dilutedn HBS. Following removal of the HBS medium, fresh HBS

edium was added to the cultures with or without inhibitoryompounds. Cultures were left unstimulated (control) or were

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40 M.J. Abbey et al. / Journal of Eth

timulated with KCl or capsaicin for an additional 60 min andhe amount of secreted CGRP measured. As another control,ultures were treated with equivalent amounts of the appropriateehicle (dimethylsulfoxide (DMSO) for capsaicin or methanolor Theobroma cacao extract) for 60 min and the amount ofecreted CGRP determined. To test for possible cytotoxic effectsf the various treatments, neuronal viability was assessed inrigeminal cultures 24 h after treatment using the CellTiter 96ssay following manufacturer’s instructions (Promega, Madi-on, WI). The absorbency at 490 nm was measured 2 h followingddition of the CellTiter 96 Aqueous One Solution. Each exper-mental condition was performed in duplicate and repeated int least three independent experiments. The data were reporteds mean ± S.E.M. Statistical analysis was performed usinghe Mann–Whitney U-test. Results were considered significanthen p was less than 0.05.

.5. Immunocytochemistry

Dissociated trigeminal ganglion cells were plated directly onDL (MW 30,000–70,000; Sigma)-coated 13-mm plastic cov-rslips equivalent to one ganglion per three coverslips in L15omplete medium. All cells were incubated for 48 h at 37 ◦C theninsed briefly with phosphate buffered saline (PBS) and treatedith 4% paraformaldehyde for 30 min at room temperature andith 0.2% Triton X-100 in PBS for an additional 15 min to fix

nd permeabilize the cells. Cultures were incubated overnightt 4 ◦C with chicken anti-CGRP antibodies (1:500 in PBS; Neu-omics, Edina, MN) or rabbit anti-TRPV1 antibodies (1:1000 inBS; Neuromics). Immunoreactive proteins were detected fol-

owing incubation with Rhodamine Red X-conjugated donkeynti-chicken or FITC-conjugated donkey anti-rabbit IgG poly-lonal antibodies (1:100 in PBS, Jackson Immuno-Researchaboratories, West Grove, PA) for 1 h at room temperature.rior to viewing, cells were mounted using Vectashield mount-

ng media (Vector Laboratories, Burlingame, CA) containing′,6 diamidino-2-phenylindole (DAPI) to identify nuclei. Cellsere photographed with an Olympus DP70 camera mounted on

n Olympus BX41 fluorescent microscope and image collectionnd analysis performed using Olympus MicroSuite Five imagerocessing software (Center Valley, PA).

.6. Intracellular calcium measurements

For intracellular calcium imaging, trigeminal ganglion cul-ures enriched for neurons were plated at a density equivalento 1.5–2 ganglia per well on PDL-coated, plastic or glass cover-lips. Cells were incubated with Fura-2AM and pleuronic acidInvitrogen Inc., Eugene, OR) at final concentrations of 5 �M,sing a similar protocol as previously described (Durham andusso, 1999, 2000). Absorbance was determined at 340 nm and80 nm for 1 s at 20 s intervals in a Victor 3V plate reader fol-owing manufacturer’s instructions (Perkin-Elmer, Wellesley,

A). Basal levels were obtained for 5 min before treatmentith 90 mM KCl, 2 �M capsaicin, or 17 �g/g of extract dryeight (100 �g/ml GAE Theobroma cacao extract). After addi-

ion of stimulatory agents or Theobroma cacao, intracellular

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rmacology 115 (2008) 238–248

alcium levels were determined for an additional 20 min. Inome wells treated with Theobroma cacao extract, cultures wereubsequently treated with 90 mM KCl or 2 �M capsaicin andntracellular calcium levels measured for another 5 min. Data areeported as the ratio of the 340–380 wavelength measurementsnd, thus, are the averages of all the cells in each well. Eachxperimental condition was repeated a minimum of six times.

.7. In vivo studies

Female post-estrous rats (200–225 g) were anesthetized byntraperitoneal injection of ketamine (96 mg/kg) and xylazine7.2 mg/kg) solution (0.2–0.3 ml; Sigma). Initially, the retro-rade tracer True Blue (25 �l of 2 mg/ml in deiononized H2O;iotium, Inc., Hayward, CA) was injected into each TMJapsule. After 5–7 days, the animal was sacrificed and bothrigeminal ganglion removed and immediately placed in Neg0 mounting medium (Perkin-Elmer, Downers Grove, IL) at25 ◦C for further analysis. For immunohistochemical studies,

0 �m sections were fixed, permeabilized, and incubated withnti-CGRP (1:500 in PBS; Neuromics) and/or TRPV1 antibod-es (1:1000 in PBS; Neuromics) at 4 ◦C for 24 h. Immunoreactiveroteins were visualized following incubation with fluorescentlyabeled secondary antibodies as described above. In some stud-es, multiple image analysis was performed to allow observationf the retrograde tracer True Blue and expression of CGRP andapsaicin receptor TRPV1 in the entire ganglion. For studiesn CGRP levels in trigeminal ganglion, animals were eithereft untreated or both TMJ capsules were injected with cap-aicin (25 �l of 10 �M solution) or vehicle (DMSO) 2 h prioro harvesting the ganglion. For the cocoa studies, Theobromaacao extract (25 �l; 424 �g/g or 638 �g GAE) or vehicle (25 �lethanol) was injected into both capsules 24 h prior to injection

f capsaicin. Following treatment, the animals were sacrificednd the posterior third of each ganglion, which corresponds tohe mandibular or V3 branch, was removed, placed in HBSH 7.4, homogenized, centrifuged, and the amount of CGRPn the supernatant determined by radioimmunoassay. The totalmount of protein in each sample was determined by the Brad-ord method (Bio-Rad Laboratories, Hercules, CA).

.8. Statistical analysis

The data are reported as mean (pmol/mg total pro-ein) ± S.E.M. Each experimental condition was repeated in ateast three independent experiments. Statistical analysis was per-ormed using the nonparametric Mann–Whitney U-test. Resultsere considered significant when p was less than 0.05. All sta-

istical tests were preformed using Minitab Statistical Software,elease 14.

. Results

.1. Theobroma cacao extract represses stimulated CGRP

ecretion

Primary rat trigeminal ganglia cultures were utilized tonvestigate the effects of Theobroma cacao extract on CGRP

M.J. Abbey et al. / Journal of Ethnopharmacology 115 (2008) 238–248 241

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tincubated with the same amount of Theobroma cacao extract asdescribed above. In contrast to the effect of Theobroma cacaoon stimulated CGRP release, pretreatment with Theobromacacao did not inhibit the amount of CGRP secreted under basal,

Fig. 2. Theobroma cacao extract represses stimulated CGRP secretion. The rela-tive amount of CGRP secreted from d2 trigeminal neurons in 1 h from untreatedcontrol cultures (CON), cultures treated with 90 mM KCl or 2 �M capsaicin(CAP), or cultures pretreated for 30 min with increasing amounts of Theobromacacao extract. The final amount of Theobroma cacao extract (10, 50, 100, 200)is reported as �g/ml expressed as gallic acid equivalents or GAE, which corre-

ig. 1. CGRP and TRPV1 are co-expressed in most cultured trigeminal gangln poly-d-lysine-coated coverslips. (B and D) The same culture shown in pane0 �m (C and D).

ecretion. Under our culture conditions, which are based onreviously published procedures (Durham and Russo, 1999;ellamy et al., 2006), >95% of the neurons express CGRP

Fig. 1A, 1C). The majority of the CGRP positive neurons>90% cells) also expressed the TRPV1 receptor, which iden-ifies capsaicin-responsive sensory neurons involved in painransmission (Fig. 1B, D). As a control, no staining was observedn any trigeminal ganglion cells in the absence of primary anti-odies (data not shown). These data demonstrate that under ourulture conditions the majority of trigeminal neurons expressoth CGRP and TRPV1.

To determine the effect of Theobroma cacao extract on stim-lated release of CGRP from trigeminal neurons, cultures wereretreated for 30 min with Theobroma cacao extract prior toddition of KCl or capsaicin and the amount of CGRP secretedn 1 h measured. As seen in Fig. 2, stimulation with 90 mM KClaused an almost 8-fold increase in CGRP release while treat-ent with 2 �M capsaicin caused a 5-fold increase. Pretreatmentith increasing amounts of Theobroma cacao extract resulted

n a significant reduction in the amount of CGRP secretedn response to KCl. Since not much difference was observedetween the 8.5, 17, or 34 �g/g of Theobroma cacao extract dryeight (50, 100, and 200 �g/ml GAE), 17 �g/g or 100 �g/ml

AE was used in all subsequent experiments. At this concentra-

ion of Theobroma cacao extract, capsaicin stimulation of CGRPelease was also greatly reduced to a level similar to the effecteen on KCl stimulation.

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urons. (A and C) CGRP expression in d2 trigeminal ganglion neurons platedC costained for TRPV1 expression. Magnification bar = 200 �m (A and B) or

To determine whether Theobroma cacao extract could inhibithe basal amount of CGRP secreted, unstimulated cultures were

ponds to 1.7, 8.5, 17, or 34 �g/g of extract dry weight. The mean basal rate ofGRP release was 16.0 pg/h/well ± S.E.M. 0.685. The secretion rate for eachondition (n > 6) was normalized to the basal rate for each well. + = p < 0.01hen compared to control, * = p < 0.001 when compared with KCl stimulated

evels, # = p < 0.001 when compared to capsaicin-stimulated levels.

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nstimulated conditions (data not shown). In fact, at the higheroncentrations (17 and 34 �g/g; 100 and 200 �g/ml GAE),heobroma cacao extract slightly, yet significantly (p < 0.01),timulated CGRP secretion (∼2-fold, n = 12). As a control, cul-ures were also treated for equal times with equivalent volumesf vehicle for Theobroma cacao extract (methanol) or capsaicinDMSO). Secreted CGRP levels in the methanol or DMSOreated samples were not significantly different than those forhe control samples (n = 10; p < 0.45 for both conditions). Tostablish that the inhibitory effect of Theobroma cacao was notue to cytotoxicity, neuronal viability was determined 24 h afterreatments. Based on data from the CellTiter 96 assay (n = 3), noignificant difference in neuronal viability was observed follow-ng treatment with Theobroma cacao extract or methanol whenompared to untreated cultures. Taken together, these resultsemonstrate that Theobroma cacao extract can repress stimu-ated but not basal CGRP secretion, and the inhibitory effect isot due to cytotoxicity.

.2. Theobroma cacao blocks KCl-mediated increase in

ntracellular calcium

Having shown that Theobroma cacao extract could inhibitGRP release in response to KCl and capsaicin, calcium imag-

onal

ig. 3. Theobroma cacao extract inhibits KCl and capsaicin-mediated increases in irigeminal ganglion neurons in response to 90 mM KCl (A) or 2 �m capsaicin (C),xtract dry weight) prior to addition of KCl (B) or capsaicin (D). The levels are the avxperiments is shown for each experimental condition. All data are expressed relative

rmacology 115 (2008) 238–248

ng was used to determine whether the inhibitory effect ofheobroma cacao on stimulated CGRP secretion involved inhi-ition of increased intracellular calcium levels. Basal calciumevels were measured in HBS medium prior to stimulation with0 mM KCl or 2 �M capsaicin. Both KCl and capsaicin treat-ent alone caused a rapid increase in intracellular calcium

bove basal levels as shown in representative calcium plotsFig. 3A and C). The mean and S.E.M. at the highest cal-ium level for KCl was 1.20 ± 0.02 while for capsaicin theevel was 1.19 ± 0.02 relative units (n = 6). However, treatmentith Theobroma cacao extract caused a prolonged decrease inasal intracellular calcium levels and prevented the typical cal-ium increase in response to KCl (Fig. 3B) or capsaicin aseen in representative calcium plots (Fig. 3D). The mean and.E.M. following the addition of Theobroma cacao prior toCl treatment was 0.68 ± 0.05, while after KCl treatment theean level was 0.69 ± 0.06. Similarly, the mean and S.E.M.

ollowing the addition of Theobroma cacao prior to capsaicinreatment was 0.75 ± 0.03, while after capsaicin treatment was.75 ± 0.03. Based on these data, Theobroma cacao repression

f KCl or capsaicin-stimulated CGRP release from trigeminaleurons is likely to involve inhibition of calcium channel activitynd blocking the subsequent elevation of intracellular calciumevels.

ntracellular calcium levels. Relative intracellular calcium levels in d2 culturedor pretreated with Theobroma cacao extract (100 �g/ml GAE or 17 �g/g oferage of all the cells in each well. A representative graph from >6 independentto basal calcium levels, which were made equal to one.

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.3. Theobroma cacao repression of CGRP in an in vivoodel of TMJ inflammation

To determine whether Theobroma cacao extract could inhibitGRP expression in an in vivo model of TMJ inflammation, theffect of Theobroma cacao on capsaicin-stimulated CGRP lev-ls in the trigeminal ganglion was investigated. In this model,apsaicin is injected into the TMJ capsule to activate nociceptiveeurons that mediate peripheral inflammation and pain. Initially,he fluorescent dye, True Blue, was injected into the TMJ cap-ule to retrograde label neuronal cell bodies within the trigeminalanglion that provide sensory innervation of the capsule. Mul-iple image alignment was used to obtain pictures of the entireanglion including the ophthalmic (V1), maxillary (V2), andandibular (V3) regions. After 5–7 days, the dye was readily

bservable in the cell body of neurons found in clusters or bandsithin the posterior lateral region of the ganglion, which corre-

ponds to the mandibular or V3 branch (Fig. 4A). The number ofabeled cells was greatest in the middle sections of the ganglionhen compared to the most dorsal and ventral sections in which

ess than 10 cells were identified. The majority of neurons thatrovide sensory innervation of the TMJ capsule were found toxpress both CGRP (Fig. 4C) and TRPV1 (Fig. 4D).

Immunohistochemistry was used to study the effect of Theo-roma cacao extract on CGRP-IR levels within the trigeminalanglion of untreated animals or animals injected with capsaicinn the TMJ capsule. In untreated animals, CGRP was readilyetected in the cell bodies of neuronal cells in the mandibularegion of the ganglion (Fig. 5A). In contrast, the levels of CGRPithin this region of the ganglion were greatly diminished afterh following capsaicin injection in the TMJ capsule (Fig. 5B).he decrease in immunoreactive CGRP is likely caused by the

elease of CGRP from the terminals and cell body of neuronsn response to capsaicin stimulation, which has been previouslyeported to cause neuropeptide release (Jansen-Olesen et al.,996; Garry et al., 2000). Significantly, the stimulatory effectf capsaicin on CGRP-IR levels in the ganglion was preventedn animals in which Theobroma cacao extract was injected intohe TMJ capsule 24 h prior to capsaicin stimulation (Fig. 5D). Inheobroma cacao treated animals, the level of CGRP-IR within

he ganglion was returned to levels observed in untreated, con-rol animals. This in vivo finding is in agreement with our initro results in which Theobroma cacao repressed capsaicintimulation of CGRP secretion from cultured trigeminal neu-ons (Fig. 2). In contrast, overnight treatment with Theobromaacao did not appear to affect CGRP-IR levels in the mandibu-ar region of the ganglion, since levels were comparable to thoseeen in untreated animals (Fig. 5C).

As a complementary approach to demonstrate thatheobroma cacao can repress the stimulatory effects ofapsaicin-mediated trigeminal nerve activation, CGRP levelsithin the ganglion were determined by radioimmunoassay.he amount of CGRP is reported as pmol/mg of total pro-

ein. As seen in Fig. 6, capsaicin stimulation for 2 h causedsignificant decrease in CGRP levels within the mandibu-

ar region of the ganglion when compared to control levels.his stimulatory effect was completely blocked by pretreatment

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rmacology 115 (2008) 238–248 243

ith Theobroma cacao extract 24 h prior to capsaicin injec-ion. Injection of DMSO (capsaicin vehicle) for 2 h or methanolTheobroma cacao vehicle) for 24 h did not cause any signifi-ant change in CGRP levels compared to control values. Theseesults, which are in agreement with our immunohistochemicalndings, demonstrate that Theobroma cacao extract can repressapsaicin-stimulated CGRP release from trigeminal nerves thatrovide sensory innervation of the TMJ capsule.

. Discussion

Based on epidemiological studies, populations that consumeoods and drinks rich in polyphenols have a lower incidencef chronic inflammatory diseases such as arthritis, atheroscle-osis, and chronic heart failure (Stoclet et al., 2004; Goggs etl., 2005; Buijsse et al., 2006; McCullough et al., 2006). Theeneficial effects of polyphenols in cardiovascular disease haveeen shown to involve inhibition of key enzymes and transcrip-ion factors that are known to promote inflammation (Bode andigang, 2003; Yoon and Baek, 2005). Much of this research has

ocused on the antioxidant and radical scavenging capacity ofolyphenols in immune and endothelial cells to suppress inflam-ation (Sanbongi et al., 1998; Stoclet et al., 1999; Kempuraj et

l., 2005). Furthermore, many of the health benefits attributedo dietary polyphenols are due to their ability to repress inflam-

atory responses by inhibiting the production and release ofytokines and other inflammatory mediators from immune cellsChen et al., 2002; Veronique et al., 2004; Wheeler et al., 2004).owever, the effect of polyphenols on sensory neuron func-

ion and excitability is not well understood. Towards this end,e investigated whether biologically active compounds isolated

rom Theobroma cacao beans, which contain higher levels ofolyphenols than red wine or green tea, could inhibit CGRPxpression in cultured trigeminal ganglia neurons and in vivoodel of TMJ inflammation.In this study, we showed that the stimulatory effect of KCl and

apsaicin on CGRP secretion from trigeminal ganglion neuronsas significantly repressed by pretreatment with Theobroma

acao extract enriched for polyphenols. The concentration ofolyphenols in our Theobroma cacao extract, which was foundo suppress neuronal activity and was not toxic to neuronal cells,as similar to concentrations reported to regulate gene expres-

ion in non-neuronal cells (Noe et al., 2004; Ramiro et al., 2005).hile KCl is known to activate neurons via chemical depolariza-

ion, capsaicin selectively excites nociceptive C and A� fibers toause neuropeptide release (Caterina et al., 1997). Based on ouralcium imaging studies, we speculate that Theobroma cacaoepression of trigeminal neuron activity involves blockage ofhe normal physiological increase in intracellular calcium lev-ls in response to KCl and capsaicin. It is well established thathe cellular effects of KCl and capsaicin can be mediated byctivation of calcium channels (Bell, 1993; Kopanitsa et al.,995; Brosenitsch et al., 1998; Mohri et al., 2003). Our finding

s in agreement with a recent study that reported an inhibitoryffect of the flavonols, a class of plant polyphenols, on stimulatednterleukin secretion which involved suppression of intracellu-ar calcium levels in cultured mast cells (Kempuraj et al., 2005).

244 M.J. Abbey et al. / Journal of Ethnopharmacology 115 (2008) 238–248

Fig. 4. Expression of CGRP and TRPV1 in trigeminal ganglion neurons providing innervation of the TMJ capsule. (A) Cellular localization of True Blue in neuronalc r injecC to thn

Hefi

ells within the mandibular or V3 region of the trigeminal ganglion 5 days afteolocalization of CGRP (C) and TRPV1 (D) in neurons providing innervationuclear dye DAPI to identify nuclei of both neuronal and glial cells (B).

owever, our data provide the first evidence, to our knowl-dge, of Theobroma cacao extract blocking neuropeptide releaserom sensory neurons via a mechanism that is likely to involvenhibition of calcium channel activity.

tfi

tion of the dye into the TMJ capsule. The entire trigeminal ganglion is shown.e TMJ capsule. The same section of trigeminal ganglion was stained with the

Previously, we had reported that the anti-migraine drug suma-riptan could inhibit stimulated but not basal release of CGRProm trigeminal neurons (Durham and Russo, 1999). Similarly,n this study, we found that Theobroma cacao did not inhibit

M.J. Abbey et al. / Journal of Ethnopharmacology 115 (2008) 238–248 245

Fig. 5. Theobroma cacao extract blocks effect of capsaicin on CGRP levels in vivo in trigeminal ganglion. CGRP-IR expression in the mandibular region of trigeminalg als inji showi

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anglion sections (40 �m) obtained from control, untreated animals (A), animnjected with Theobroma cacao extract 24 h prior to capsaicin injection (D) ismage alignment of the entire trigeminal ganglion is shown.

asal CGRP secretion but rather, only repressed stimulatedGRP release. Interestingly, while both sumatriptan and Theo-roma cacao extract were shown to regulate CGRP secretionrom cultured trigeminal ganglion neurons in a similar man-er, the mechanism of repression appears to be quite distinct.

umatriptan is a member of the triptan class of serotonergicrugs which are commonly used to abort the pain associatedith migraine headache (Dodick and Martin, 2004). Relief ofigraine headache with sumatriptan has been shown to corre-

eept

ected with capsaicin for 2 h (B) or Theobroma cacao for 24 h (C), or animalsn. Only the mandibular or V3 region of each ganglion obtained from multiple

ate with a return of CGRP to normal levels (Buzzi et al., 1991;oadsby and Edvinsson, 1991, 1993). Sumatriptan repressionf KCl-stimulated CGRP release involves a sustained elevationf intracellular calcium and activation of specific phosphatasesDurham and Russo, 1999). In contrast, Theobroma cacao

xtract caused a prolonged decrease in intracellular calcium lev-ls, a cellular event not seen with sumatriptan. Furthermore,retreatment with Theobroma cacao prevented the normal rapidransient increase in intracellular calcium in response to KCl

246 M.J. Abbey et al. / Journal of Ethnopha

Fig. 6. Theobroma cacao extract represses stimulatory effect of capsaicin onCGRP levels in trigeminal ganglion. The amount of CGRP was determined byradioimmunoassay in trigeminal ganglion obtained from control, untreated ani-mals (CON, n = 13) or animals treated for 2 h with DMSO (n = 4) or capsaicin(CAP, n = 7). CGRP levels were also measured in ganglion obtained from ani-mals 24 h after injection of methanol (MET, n = 4) or from animals initiallyihc

arfs

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icTrTwtiIndcmweiMtctpftalibF

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njected with Theobroma cacao for 24 h and then with capsaicin 2 h prior toarvesting (n = 4). * p < 0.005 when compared to control, # p < 0.001 whenompared to capsaicin-stimulated levels.

nd capsaicin. Thus, it appears that Theobroma cacao extractepresses CGRP secretion from trigeminal neurons by a dif-erent mechanism than reported for the migraine abortive drugumatriptan.

CGRP is believed to play an important role in the genesisnd maintenance of inflammation and pain associated with bothigraine and TMJ pathology (Lassen et al., 2002; Juhasz et al.,

003; Lobbezoo et al., 2004). In this study, we used an in vivoodel of TMJ inflammation to investigate the effect of Theo-

roma cacao extract on trigeminal nerve activation and CGRPelease. Initially, we used a retrograde tracer to identify neuronshat provide sensory innervation to the TMJ capsule within theosterior lateral region of the trigeminal ganglion. Neuronal cellodies were arranged in discrete bands within the mandibularegion of the ganglion as reported in a previous study (Takedat al., 2005). We found that the majority of the neurons provid-ng innervation of the TMJ capsule co-expressed CGRP and theRPV1 receptor. Similarly, others (Ichikawa et al., 2004; Loit al., 2006) have reported TRPV1 and CGRP co-expressionn nerves providing innervation of cells lining the synoviumf the TMJ capsule. TRPV1 receptors, which are activatedy capsaicin, heat, and protons, are abundantly expressed bynmyelinated C-fibers involved in pain transmission (Caterinat al., 1997). In addition, activation of TRPV1 receptors has beenhown to cause CGRP release from sensory neurons (Carlesont al., 1997; Spears et al., 1998; Gibbs et al., 2006). Peripheralelease of CGRP promotes inflammation within the joint (Spearst al., 2005), while its release centrally in the trigeminal nucleusaudalis is correlated with increased pain perceptions and canead to central sensitization and allodynia (Hutchins et al., 2000;enkins et al., 2004). In this study, injection of capsaicin into theMJ capsule was shown to cause a decrease in CGRP levels

n trigeminal neurons within the mandibular or V3 region ofhe ganglion. This decrease was likely due to release of CGRProm nerve terminals as well as from the cell body of the neu-ons. Our capsaicin findings are in agreement with other studies

R

A

rmacology 115 (2008) 238–248

n which CGRP levels in the trigeminal ganglion of capsaicinreated animals were found to be lower than levels in controlnimals (Spears et al., 1998).

Having demonstrated that Theobroma cacao extract couldnhibit KCl- and capsaicin-stimulated CGRP secretion fromultured trigeminal neurons, we wanted to determine whetherheobroma cacao could block capsaicin-stimulated CGRPelease from trigeminal ganglion neurons in vivo. Injection ofheobroma cacao extract 24 h prior to capsaicin administrationas shown to effectively block changes in CGRP levels within

he ganglion when compared to untreated control animals. Thus,mportantly, our in vivo findings agree with our in vitro studies.n another in vivo study, a cocoa extract was shown to sig-ificantly decrease NO release from macrophages as well asown-regulate mRNA expression of inflammatory cytokines andhemokines (Ramiro et al., 2005). It will be of interest to deter-ine whether Theobroma cacao extract enriched in polyphenolsill decrease CGRP gene expression. Repression of CGRP

xpression may be clinically important given the role of CGRPn inflammatory and neuropathic pain (van Rossum et al., 1997;

a and Quirion, 2006). For example, CGRP has been reportedo play an important role in the initiation and maintenance ofapsaicin-induced secondary allodynia and hyperalgesia and,hus, in the development of central sensitization in response toeripheral inflammation (Sun et al., 2003). In addition, resultsrom in vivo studies have demonstrated that CGRP contributeso nociceptive behavior associated with inflammation (Han etl., 2005; Ambalavanar et al., 2006). Based on our findings, it isikely that repression of stimulated CGRP expression by biolog-cally active compounds isolated from Theobroma cacao woulde beneficial in treating TMJ disorders, for which there are noDA approved pharmaceutical treatments.

In summary, results from this study have shown that CGRPecretion from stimulated trigeminal neurons can be directlynhibited by a methanol extract of Theobroma cacao beans. Sig-ificantly, it was shown that Theobroma cacao extract couldlock CGRP release from cultured trigeminal neurons as well asn an in vivo model of TMJ inflammation. Based on our calciummaging data, the inhibitory effect of Theobroma cacao extractn trigeminal neurons likely involves blockage of calciumhannel activity. Thus, our findings demonstrate that Theo-roma cacao extract contains biologically active compoundshat repress trigeminal nerve activation and, hence, release ofGRP, which is known to contribute to inflammation and painithin the TMJ. Furthermore, our results provide evidence that

he medical benefits attributed to the use of cocoa in ancientayan and Aztec cultures for the treatment of tooth and joint ail-ents may have involved repression of sensory nerve activation.

cknowledgement

The authors would like to thank Ryan Cady for his technicalssistance.

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